PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3^-/- mouse embryo fibroblasts (Pdia3^-/- MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3^-/- MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WT-MEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3^-/- MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3^-/- MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3^-/- MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target.